The overall objective of the project is to examine regulatory mechanisms governing the formation and activity of glucose degrading enzymes in the highly versatile bacterium Pseudomonas cepacia (P. multivorans). The work has focused on the unusual occurrence of multiple glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGAD) species in this bacterium and on the joint loss of G6PD and 6PGAD species in mutants selected for failure to grow normally on glucose. We previously purified the G6PD and 6PGAD species active with NAD and subject to inhibition by ATP and NADPH, and characterized them with respect to subunit composition and other properties. The NADP-specific 6PGAD, which is insensitive to inhibition by ATP and NADPH, was also purified and characterized. Purified preparations of the NADP-specific G6PD were not obtained due to problems of enzyme instability. The results indicated that the joint loss of G6PD and 6PGAD in the mutants was not due to all 4 enzymes sharing a common subunit or to regulatory defects requiring coordinate regulation of all 4 enzymes. Our recent efforts have concentrated on purification and characterization of the NADP-specific G6PD. We have used our initial observation that the enzyme increases in apparent molecular weight in the presence of G6P to develop a purification scheme based on size fractionation. The enzyme has been purified to homogeneity and we are studying its subunit composition, its antigenic properties, and ligand dependent changes in its sedimentation properties. Other aspects of the studies include (1) characterization of mutants with altered glucose catabolism (2) studies of glutathione metabolism as it relates to glucose catabolism and 3) development of a transduction system to permit genetic analysis of various mutations affecting glucose catabolism and isoleucine-valine metabolism in P. cepacia.